AGGF1调控AMPK-mTOR-ULK1信号通路促进视网膜血管生成机制研究  

Effects of angiogenic factor with G patch and FHA domains 1 on angiogenesis of human retinal microvascular endothelial cells and its action mechanism in vitro

作　　者：李蓉[1] 姚国敏[1] 周凌霄[1] 闫瑾 张敏[2] 李亚[2] LI Rong;YAO Guomin;ZHOU Lingxiao(Department of Ophtalmology,The First Hospital Affiliated to Xi’an Medical College,Shaanxi,Xi’an 710077,China)

机构地区：[1]西安医学院第一附属医院眼科,西安市710077 [2]西安医学院第一附属医院内分泌科,西安市710077 [3]西安医学院医学技术学院

出　　处：《河北医药》2022年第22期3365-3369,共5页Hebei Medical Journal

基　　金：陕西省科技厅社发基金项目(编号:2021SF-157);陕西省科技厅自然科学基金项目(编号:2019JM-514)。

摘　　要：目的研究G补缀FHA域血管生成因子1(AGGF1)对人视网膜血管内皮细胞(HRMECs)血管生成的影响及机制。方法将体外培养的HRMECs随机分为对照组、AGGF1组和AMPK信号通路抑制剂Compound C+AGGF1组,对照组细胞在M199培养基中培养,AGGF1组细胞在M199培养基中加入0.5μg/ml AGGF1进行培养,Compound C+AGGF1组在M199培养基中加入5μmol/L Compound C及0.5μg/ml AGGF1进行培养。培养48 h,分别采用Transwell及Matrigel法检测细胞迁移和管腔形成,采用Western blotting法检测各组细胞的自噬关键信号通路蛋白AMPK、mTOR和ULK1的表达,采用可表达GFP-LC3B融合蛋白的质粒转染细胞,荧光显微镜下观察细胞内自噬的变化。结果AGGF1组中细胞迁移数和管腔形成数均明显高于对照组和Compound C+AGGF1组(均P<0.001),Compound C+AGGF1组细胞迁移数和管腔形成数均高于对照组,差异均有统计学意义(P<0.01)。对照组、AGGF1组和Compound C+AGGF1组细胞中的AMPK、mTOR和ULK1蛋白表达差异均无统计学意义(P>0.05)。AGGF1组细胞中p-AMPK和p-ULK1值均明显高于对照组和Compound C+AGGF1组,差异均有统计学意义(P<0.01),AGGF1组细胞中p-mTOR值均明显低于对照组和Compound C+AGGF1组,差异均有统计学意义(P<0.05);Compound C+AGGF1组的p-AMPK和p-ULK1值均高于对照组(P<0.01),Compound C+AGGF1组的p-mTOR值低于对照组,差异有统计学意义(P<0.001)。对照组细胞中出现较弱的GFP-LC3B绿色荧光,AGGF1组绿色荧光明显强于对照组和Compound C+AGGF1组,自噬增强(P<0.001);Compound C+AGGF1组的绿色荧光强于对照组(P<0.05)。结论AGGF1通过激活AMPK-mTOR-ULK1自噬信号通路促进视网膜微血管内皮细胞的血管生成。Objective To investigate effects of angiogenic factor with G patch and FHA domains 1(AGGF1)on angiogenesis of human retinal microvascular endothelial cells(HRMECs)and its action mechanism in vitro.Methods HRMECs cultured in vitro were randomly divided into control group,AGGF1 group and Compound C+AGGF1 group.The cells in control group were cultured in M199,and the cells in AGGF1 grou were cultured in M199 medium adding 0.5μg/ml AGGF1,and the cells in Compound C+AGGF1 group were treated with 5μmol/L Compound C and 0.5μg/ml AGGF1 in M199 medium.After 48-hour culture,transwell and matrigel assay was used to detect the cellular migration and tube formation,respectively;Western Blot was performed to detect the expression levels of key factors in autophagy signaling pathway in different groups,including AMPK,mTOR and ULK1.And HRMECs were transfected with GFP-LC3B and the changes of autophagy were observed under fluorescent microscope.Results The number of migrated cells and tube formation number in AGGF1 group were significantly higher than those in control group and Compound C+AGGF1 group(P<0.01),moreover,which in Compound C+AGGF1 group were significantly higher than those in control group(P<0.01).The levels of p-AMPK and p-ULK1 in AGGF1 group were significantly higher than those in control group and Compound C+AGGF1 group(P<0.01),however,the levels of p-mTOR were significantly lower than those in control group and Compound C+AGGF1 group(P<0.05).Moreover the levels of p-AMPK and p-ULK1 in Compound C+AGGF1 group were significantly higher than those in control group(P<0.01),however,the levels of p-mTOR were significantly lower than those in control group(P<0.05).In addition the weak green fluorescence of GFP-LC3B was observed in control group,and the fluorescence intensity in AGGF1 group was significantly higher than that in control group and Compound C+AGGF1 group(P<0.01),moreover,which in Compound C+AGGF1group was significantly higher than that in control group(P<0.05).Conclusion AGGF1 can promote the angiogenesis

关 键 词：AGGF1 自噬 AMPK-mTOR-ULK1 血管生成 视网膜血管内皮细胞 

分 类 号：R774.1[医药卫生—眼科]